The recent approval of anti-PD-L1 immunotherapy in combination with nAB-paclitaxel for metastatic triple-negative breast cancer (TNBC) highlights the need to understand the role of chemotherapy in modulating the tumor-immune microenvironment (TIME). Patients with TNBC are routinely treated with neoadjuvant chemotherapy (NAC). Stromal tumor-infiltrating lymphocytes (sTILs) in the pre-treatment diagnostic biopsy are predictive of pathologic complete response (pCR). In patients with residual disease (RD) at surgery, sTILs confer good prognosis. However, the effect of chemotherapy on sTILs and how it influences the TIME are poorly understood. We examined immune-gene expression patterns before and after NAC in a series of 83 breast tumors, including 44 TNBCs, from patients with RD. sTILs were enumerated by standardized guidelines. Gene expression patterns were tested for association with recurrence-free (RFS) and overall survival (OS). T cell receptor sequencing (TCRseq) was performed on a subset (n=15) of tumors. In 4 patients undergoing NAC, PD-1-high and -negative CD8+ peripheral blood mononuclear cells (PBMCs) were profiled using single-cell RNAseq and multiplexed cytokine secretion assays. Post-NAC sTILs (≥30%) were only predictive of outcome (RFS p=0.019; OS p=0.05) in TNBC patients, but not in non-TNBC patients (RFS p=0.28; OS p=0.78) confirming that the prognostic capacity of sTILs is confined to TNBC. Pre-NAC sTILs were not predictive of outcome in either group, likely due to exclusion of patients experiencing pCR. The change in sTILs during NAC did not prognosticate outcome in TNBC, suggesting that in the post-NAC setting, only the most proximal measurement of sTILs is meaningful. However, these results did suggest that NAC alters the TIME. To examine the interplay among NAC, the TIME, and clinical outcomes, we tested the change in expression of 770 immune-related genes during NAC in univariate cox-proportional hazards models. In non-TNBC, no change in expression of any single gene was associated with RFS or OS at a false-discovery rate (FDR) of 10%. In TNBC, individual changes in 12 genes and 204 genes were identified as associated with RFS and OS, respectively (FDR<10%). Interestingly, in nearly all cases, upregulation of these genes during NAC was associated with improved outcome, with only 1 and 15 genes being associated with poor RFS and OS, respectively. Collapsing genes to functional and cell-type specific signatures gave similar insights: T cell, NK cell, TNF-superfamily, and toll-like receptor signatures were highly prognostic. Surprisingly, NAC did not alter T cell clonality in TNBC. Thus, the immunologic impact of chemotherapy appears to be specific to TNBC and is primarily a beneficial effect but does not appear to appreciably expand the clonality of tumor-infiltrating T cells. Using fresh PD-1HI CD8+ T cells isolated from PBMCs of patients undergoing NAC, we detected a significant increase in cytolytic and inflammatory cytokines secreted in 2 TNBC patients after chemotherapy, but not in 2 non-TNBC patients, which was particularly dramatic in one TNBC patient who experienced a pCR. A further characterization of PD-1HI CD8+ cells by single-cell RNAseq identified a sizeable expansion of cytolytic gene (granulysin, Ksp37, granzyme) expressing cells in the TNBC patient with pCR compared to the TNBC patient with RD. In conclusion, we have characterized the effects of NAC on the TIME. TNBC appears to be uniquely sensitive to the immunologic effects of NAC, and most of these effects are primarily stimulatory, rather than repressive. Finally, these changes can be observed in the PD-1HI CD8+ peripheral T cell compartment and appeared to co-occur with pCR.

Citation Format: Justin M Balko, Mellissa Nixon, Paula I Gonzalez-Ericsson, Mark A Pilkinton, Wyatt J McDonnell, Violeta Sanchez, Susan R Opalenik, Sherene Loi, Brent Rexer, Vandana Abramson, Valerie Jansen, Simon Mallal, Jonathan D Marotti, Kevin Shee, Todd W Miller, Melinda E Sanders, Ingrid A Mayer, Roberto Salgado. Immunologic correlates of long-term outcome in the residual disease of triple-negative breast cancer after neoadjuvant chemotherapy [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-08-15.